Industrial application of glucose isomerase
In industrial starch degradation enzymes play an important role. The enzyme .alpha.-amylase is used for liquefaction of starch into dextrins with a polymerization degree of about 7-10. Subsequently the enzyme .alpha.-amyloglucosidase is used for saccharification which results in a syrup containing 92-96% glucose. The reversible isomerization of glucose into fructose is catalyzed by the enzyme glucose (or xylose) isomerase. The correct nomenclature of this enzyme is D-xylose-ketol-isomerase (EC 5.3.1.5) due to the enzyme's preference for xylose. However, because of the enzyme's major application in the conversion of glucose to fructose it is commonly called glucose isomerase. The equilibrium constant for this isomerization is close to unity so under optimal process conditions about 50% of the glucose is converted- The equilibrium mixture of glucose and fructose is known as high fructose syrup.
Fructose is much sweeter to the human taste than glucose or sucrose which makes it an economically competitive sugar substitute.
Many microorganisms which were found to produce glucose isomerase, have been applied industrially. A detailed review of the industrial use of glucose isomerases has been given by Wen-Pin Chen in Process Biochemistry, 15 June/July (1980) 30-41 and August/September (1980) 36-41.
The Wen-Pin Chen reference describes culture conditions for the microorganisms, as well as recovery and purification methods for the enzyme. In addition it also summarizes the properties of glucose isomerases such as the substrate specificity, temperature optima and pH optima, heat stability and metal ion requirement.
Glucose isomerase requires a bivalent cation such as Mg.sup.2+, Co.sup.2+, Mn.sup.2+ or a combination of these cations for its catalytic activity. Determination of 3D structures of different glucose isomerases has revealed the presence of two metal ions in the monomeric unit (Farber et al., Protein Eng. 1 (1987) 459-466; Rey et al., Proteins 4 (1987) 165-172; Henrick et al., Protein Eng. 1 (1987) 467-475).
Apart from a role in the catalytic mechanism, bivalent cations are also reported to increase the thermostability of some glucose isomerases (M. Callens et al. in Enzyme Microb. Technol. 1988 (10), 695-700). Furthermore, the catalytic activity of glucose isomerase is severely inhibited by Ag.sup.+, Hg.sup.2+, Ca.sup.2+, Zn.sup.2+ and Ca.sup.2+.
Glucose isomerases usually have their pH optimum between 7.0 and 9.0. There are several reasons why it would be beneficial to use glucose isomerase at a lower pH value. Three of these reasons;
a) stability of the sugar molecules,
b) adaptation both to previous and/or later process steps and
c) stability of the enzyme, will be further described below to illustrate this.
a) Under alkaline conditions and at elevated temperatures the formation of coloured by-products and the production of a non-metabolizable sugar (D-Psicose) are a problem. The desired working pH should be around 6.0. Around this pH degradation of glucose and fructose would be minimal.
b) A lowered pH optimum is also desirable for glucose isomerase when this enzyme is to be used in combination with other enzymes, or between other enzymatic steps, for example in the manufacturing of high fructose syrups. In this process one of the other enzymatic steps, the saccharification by .alpha.-glucoamylase is performed at pH 4.5.
c) Most of the known glucose isomerases are applied at pH 7.5. This pH value is a compromise between a higher initial activity at higher pH and a better stability of the immobilized enzyme at lower pH, resulting in an optimal productivity at the pH chosen (R. v. Tilburg, Thesis: "Engineering aspects of Biocatalysts in Industrial Starch Conversion Technology", Delftse Universitaire Pers, 1983). Application of glucose isomerase at a pH lower than 7.5 could benefit from the longer half-life and, combined with an improved higher specific activity, would consequently increase the productivity of the immobilized enzyme at that lower pH.
From the above it can be concluded that there is need for glucose isomerases with a higher activity at lower pH values under process conditions.
Many microorganisms were screened for a glucose isomerase with a lower pH optimum. Despite many efforts, this approach did not lead to novel commercial glucose isomerases.
In order to be able to change pH-activity profile of glucose isomerases towards lower pH by protein engineering it is important to recognize the underlying effects which give rise to the rapid decrease in catalytic performance at acidic pH.